gender: male age: 52 sampletype: Serum cancertype: adenocarcinoma smoke: no family_history: no tuberculosis_history: no surgery: yes designphase: Phase I molecule: Serum autoantibody proteins IgG
Extracted molecule
protein
Extraction protocol
standard protocol
Label
Cy3
Label protocol
After washing with PBST, the microarray was incubated with 1:1000 diluted Fluor conjugated goat anti-human IgG (532nm) and donkey anti-human IgM (635nm) (Jackson ImmunoResearch, West Grove, PA) at RT for 1h in the dark. After washing with PBST, the microarray was rinsed with dd H2O and dried
After washing with PBST, the microarray was incubated with 1:1000 diluted Fluor conjugated goat anti-human IgG (532nm) and donkey anti-human IgM (635nm) (Jackson ImmunoResearch, West Grove, PA) at RT for 1h in the dark. After washing with PBST, the microarray was rinsed with dd H2O and dried
Hybridization protocol
The microarray was taken out from − 80°C and then incu-bated in blocking buffer (3% BSA in PBS) at RT for 3h.Then a serum sample diluted at 1:200 in binding buffer (1%BSA in PBST) was added to the microarray and incubatedat 4°C overnight
Scan protocol
The micro-array was scanned with the LuxscanTM 10K-A (CapitalBio Corporation, Beijing, China).
Description
Phase I of the experimental design
Data processing
Data analysis was carried out using The GenePix Pro 6.0 (Axon Instruments, Foster City, CA). The signal for each spot (SNR) was defined as the ratio of the fore-ground to the background median intensity and normalized using the median value.
